Clostridium difficile toxin a induces apoptosis in leukemic cells by upregulation of endothelial il-8

IL-8 secreted from human umbilical vein endothelial cells(HUVEC) is known to induce apoptosis of myelogeneous leukemia cell line. Clostridium difficile(C. difficile) toxin A promotes IL-8 secretion from a large variety of cell types, including monocy tes and intestinal epithelial cells but such effect on endothelial cells is unknown. In this study, we investigated whether C. difficile toxin A could induce IL-8 secretion from HUVEC, and the resultant effect on apoptosis of leukemia cell lines. For the detection of IL-8 in culture media, an enzyme-linked immunosorbent assay(ELISA) system for human IL-8 was used. After 3Id passage, HUVEC were cocultured with leukemia cell lines(K562, U937, KG1; 1 x 10s cells/mL) with or without C.difficile toxin A(lmg/ml) in 24-well plates. After 2 days, the supernatants were collected by centrifugation at 15,000 rpm and were applied to the ELIS A kit. Leukemia cells and supernatants were collected for the detection of apoptosis and 1L8 level, respectively. Cell counting, Wright-Giemsa staining, and Annexin V-PI apoptosis kit were used for the detection of apoptosis and enzyme-linked immunosorbent assay(ELISA) system for human IL-8 was used for the detection of IL-8 in supernatants. As a control, leukemia cell lines were cultured with HUVEC. All experiments were performed triplicate. The levels of IL-8 in supernatants of each cell lines were higher when C.difficile toxin A was added; 65.4 ±4.8 ng/ml to 80.7 ±5.2 ng/ml(in K562), 59.6 ±3.3 ng/ml to 76.2 ±4.6 ng/ml(in U937), 70.6 ±2.4 ng/ml to 85.7 ±6.1 ng/ml(in KG 1 ). The levels of apoptosis were also increased in samples cocultured with C.difficile toxin A; 21.4 ±1.7% to 40.2 ± 2.1 %(in K562), 20.6 ±2.4% to 49.6 ±2.9%(in U937), 22.7 ±1.9% to 47.6 ±3.1 %(in KG1 ). When leukemia cell lines cultured only with C.difficile toxin A in the absence of HUVEC, the levels of IL-8 in supernatants and the levels of apoptosis of each cell lines showed similar results to the samples cocultured without C.difficile toxin A. This study showed that C. difficile toxin A could induce apoptosis against leukemic cells, possibly by promoting secretion of IL-8 from vascular endothelium.